Bacterial diversity, organic pollutants and their metabolites in two aeration lagoons of common effluent treatment plant (CETP) during the degradation and detoxification of tannery

Bioresource Technology 102 (2011) 2333–2341

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Bioresource Technology

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Bacterial diversity, organic pollutants and their metabolites in two aerationlagoons of common effluent treatment plant (CETP) during the degradationand detoxification of tannery wastewater

Ram Chandra a,⇑, Ram Naresh Bharagava a, Atya Kapley b, Hemant J. Purohit b

a Environmental Microbiology Section, Indian Institute of Toxicology Research (CSIR), Post Box 80, M.G. Marg, Lucknow 226 001, UP, Indiab Environmental Genomics Unit, National Environmental Engineering Research Institute (CSIR), Nehru Marg, Nagpur 440020, Maharashtra, India

a r t i c l e i n f o

Article history:Received 24 July 2010Received in revised form 16 October 2010Accepted 20 October 2010Available online 23 October 2010

Keywords:Tannery wastewaterBacterial diversityOrganic pollutantsBiodegradationMetabolites

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.10.087

⇑ Corresponding author. Tel.: +91 522 2476051/2472228471.

E-mail address: [email protected]

a b s t r a c t

In this study, PCR-RFLP and GC–MS approaches were used to characterize the bacterial diversity, organicpollutants and metabolites during the tannery wastewater treatment process at common effluenttreatment plant (CETP). Results revealed that the bacterial communities growing in aeration lagoon-Iwere dominated with Escherichia sp., Stenotrophomonas sp., Bacillus sp. and Cronobacter sp. while thatof aeration lagoon-II prevailed with Stenotrophomonas sp., and Burkholderiales bacterium, respectively.The HPLC and GC–MS analysis revealed that most of the organic pollutants detected in untreated tannerywastewater samples were diminished from bacterial treated tannery wastewater samples. Only twopollutants i.e. L-(+)-lactic acid and acetic acid could not be degraded by bacteria whereas benzene and2-hydroxy-3-methyl-butanoic acid was produced as new metabolites during the bacterial treatment oftannery wastewater in aeration lagoon II of CETP. Further, it was observed that after bacterial treatment,the toxicity of tannery effluent was reduced significantly allowing 90% seed germination.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Tannery industries are one of the most polluting industriesmainly causing chromium pollution in environment. There aremore than 2500 tanneries in India and most of them (nearly 80%)are engaged in chrome tanning process (Shukla et al., 2009).During the tanning process, chromium salt is used to convert hideinto leather and the wastewater containing huge amount of organ-ic matter, phenolics, tannins and heavy metals mainly chromiumas environmental pollutants is discharged into the environmentwhich causes serious soil and water pollution along with seriousthreat to human health. In environment, Cr6+ contamination altersthe structure of soil microbial communities as well as reducedtheir growth retarding the bioremediation process and if Cr6+

enters in food chain, it causes skin irritation, eardrum perforation,nasal irritation, ulceration and lung carcinoma in humans andanimals along with accumulate in placenta impairing the fetaldevelopment in mammals (Verma et al., 2001; Srinath et al.,2002; Cheung and Gu, 2007).

Microbes (bacteria/fungi) are the most important eco-friendlyagents for the degradation and detoxification of industrial pollu-tants during the biological treatment of industrial wastewaters.

ll rights reserved.

6057; fax: +91 522 2228227/

(R. Chandra).

The wastewater treatment processes carried out in aerationlagoons at common effluent treatment plant (CETP) are mostwidely accepted methods for the degradation and detoxificationof domestic as well as industrial wastewaters (Moharikar et al.,2005; Kapley et al., 2007; Moura et al., 2009). The concept of com-mon effluent treatment plant (CETP) is based on activated sludgetreatment process carried out in aeration lagoons at CETP wherea flocculent mixture of an aerobic heterogeneous population ofmicroorganisms and wastewater are aerated resulting in the con-sumption of soluble organic material and entrapment of suspendedparticulate matter by from wastewater (Munz et al., 2008). To thisCETP, the wastewater from tannery industries is transportedthrough pipelines and is collected initially in an equalization tankprior to entering the wastewater treatment plant which is specifi-cally designed and operated for the degradation and detoxificationof industrial wastewaters.

However, the microbial diversity growing in aeration lagoons ofCETP is largely depend on the nature of pollutants and geographi-cal conditions and changes in microbial diversity can compromisethe entire wastewater treatment process. Although, there are manyreports available regarding the microbial communities analysis inaeration lagoons of wastewater treatment plant (Moura et al.,2009; Kapley et al., 2007; Baker et al., 2003). But, no one hasinvestigated the microbial diversity, organic pollutants and theirmetabolites simultaneously produced during the degradation anddetoxification of tannery wastewater at CETP. To understand the

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2334 R. Chandra et al. / Bioresource Technology 102 (2011) 2333–2341

mechanism of pollutants degradation in aeration lagoons at CETP,the information about growing bacterial communities andmetabolic products is very essential. Hence, the objectives of thisstudy were to analyze the bacterial diversity, organic pollutantsand metabolites produced during the treatment process and toevaluate the environmental safety of tannery wastewater.

2. Methods

2.1. Collection of tannery wastewater from CETP

The untreated and treated tannery wastewater samples werecollected in pre-sterilized clean containers (capacity 25 l) fromthe equalization tank and two aeration lagoons of CETP, Unnao(26.48o N, 80.43o E), Uttar Pradesh, India. The characteristics andoperational conditions of the two aeration lagoons at CETP areas: capacity, 2028 KL each; organic loading rate, 910 mg l�1 for24 h a day; hydraulic retention time and temperature, 24 h atambient temperature; temperature feeding rate, ambient as re-ceived from tanneries after giving 24 h retention time in equaliza-tion tank. The wastewater treatment process in these two aerationlagoons is carried out round the clock for 365 days. However, thereis monthly slight variation in these parameters, but not signifi-cantly except dyes colours which may be vary as per orders frombuyers.

Samples were periodically collected in different seasons (i.e. insummer (May), rainy season (July), and winter season (December).The collected wastewater samples were brought to laboratory,maintained at 4 �C for the analysis of bacterial diversity, physico-chemical parameters, organic pollutants and their metabolites aswell as toxicity evaluation of tannery wastewater before and afterbacterial treatment.

2.2. Bacterial diversity analysis in two aeration lagoons of CETP

2.2.1. Total genomic DNA extraction and PCR amplification of 16SrRNA gene

For the extraction of total genomic DNA, 10 ml of wastewatersamples collected from the two aeration lagoons of CETP werecentrifuged at 5000�g for 5 min at 4 �C to pellet the bacterial cells.The bacterial pellet was washed with 5 ml of TE buffer (10 mMTris–HCl, 1 mM EDTA, pH 8.0) and re-suspended in 2 ml of TEbuffer containing 10 mg ml�1 of lysozyme followed by incubationfor 1 h at 37 �C (Henriques et al., 2006). After lyses, the lysatewas centrifuged at 15,000�g for 10 min at 4 �C and supernatantobtained was mixed with double volume of 7.5 M ammoniumacetate followed by incubation on ice for 10 min. The samples werethen centrifuged at 15,000�g for 15 min at 4 �C and an aliquot of300–500 ll of the supernatants were purified by centrifugationat 1000�g for 2 min through a spin column (Bangalore Genei,India), which was previously equilibrated and slurried in 20 mMpotassium phosphate buffer (pH 7.4).

The PCR amplification of 16S rRNA gene was performed withuniversal eubacterial primers 27F and 1492R, (Lane et al., 1991).The reaction mixture contained 5 ll of template DNA, 1 � PCR buf-fer, 10 mM of each dNTP, 3.0 mM MgCl2, 10 pmol of primer, and 2.5U of Taq DNA polymerase (Bangalore Genei, India) in a final volumeof 50 ll reaction mixture. Each PCR cycle (35 cycles in total) con-sisted of 1 min denaturation step at 94 �C, followed by 45 s anneal-ing at 55 �C, and 1.5 min elongation step at 72 �C with an initialdenaturation step at 94 �C for 5 min and a final extension step at72 �C for 15 min. The PCR products were resolved through 1.2%(w/v) agarose gel electrophoresis in 1X TAE buffer using 500 bpDNA ladder (Bangalore Genei, India) as molecular weight markerand visualized by staining with ethidium bromide. The amplified

16S rRNA gene products were gel purified by using the PCR-Clean-up kit (Bangalore Genei, India) and used in restriction diges-tion analysis.

2.2.2. Restriction digestion of amplified 16S rRNA gene and agarose gelelectrophoresis

The restriction digestion of PCR amplified 16S rRNA gene wasperformed at 37 �C for 2 h in 50 ll reaction mixture containing12 ll (�200 ng) of PCR amplified 16S rRNA gene product, 0.5 ll(5U) of restriction endonucleases (TaqI/Sau3AI), (Bangalore Genei,India), 5 ll of reaction buffer and 32.5 ll of autoclaved milli-Qwater (Coelho et al., 2003). The restriction digestion process wasrepeated twice to establish the reproducibility of results.

The restriction endonuclease (TaqI/Sau3AI) digested 16S rRNAgene products were separated through 2% (w/v) agarose gel elec-trophoresis by using Tris–borate-EDTA buffer at 75 V for 5 h. Thegels were stained with ethidium bromide (1.0 lg/ml) and DNAbands were visualized and stored by using Gel Documentation Sys-tem (Syngene, USA). 100 bp and 500 bp DNA ladder (BangaloreGenei, India) was used as molecular weight markers to determinethe molecular weight and size of DNA fragments.

2.2.3. RFLP analysis and DNA sequencingOn the basis of differences in RFLP profiles generated by TaqI

and Sau3AI restriction enzymes, nine (9) bands from aerationlagoon-I and six (6) bands from aeration lagoon-II were selectedfor sequence analysis. These bands were purified by using gelextraction kit (Bangalore Genei, India) and sequenced by usinguniversal primers M13F (50-GTAAAACGACGGCCAGT-30) andM13R (50-AACAGCTATGACCATG-30) (Shi-Yan et al., 2007) and anABI PRISM� BigDye™ Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystems, USA). The samples were analyzed in anautomatic DNA sequencer (ABI PRISM� 310 Genetic Analyzer,USA). The partial sequences obtained were subjected to BLASTsearch using the online option available at www.ncbi.nlm.nih.gov/BLAST (Altschul et al., 1997) suggesting the identity of bacte-rial communities growing in two aeration lagoons of CETP.

2.2.4. Construction of phylogenetic trees and nucleotide sequenceaccession numbers

The phylogenetic trees were constructed by using the BootstrapTree method from Clustal X software (Larkin et al., 2007). Thesequences were aligned and distances were calculated (percentdivergence) between all the pairs of sequences from multiplealignment and finally, the trees were constructed by using theBootstrap N–J method using 1000 iterations. Further, the nucleo-tide sequences were deposited in GenBank public database underthe accession numbers from FJ422465 to FJ422473 and fromFJ422474 to FJ422479 for bacterial diversity growing in aerationlagoon-I and II, respectively.

2.3. Physico-chemical analysis of tannery wastewater samples

The physico-chemical analysis of tannery wastewater sampleswas made in triplicate according to the standard methods for theexamination of water and wastewaters (APHA, 2005). The pH ofcollected wastewater samples was measured by Orion ion meter(Model-960), BOD by 5 days method, COD by open reflux method,total solids (TS) by drying method, and total nitrogen (TN) byTOC-Vcsn analyzer (Shimadzu, Japan). Phosphate and sulfate wasmeasured by vanadomolybdo-phosphoric acid colourimetric andBaCl2 precipitation methods, respectively. The concentration of dif-ferent heavy metals (Cu, Cr, Zn, Fe, Ni, Mn and Pb) was measuredusing inductively coupled plasma spectrophotometer (ThermoElectron; Model IRIS Intrepid II XDL, USA).

http://www.ncbi.nlm.nih.gov/BLAST

http://www.ncbi.nlm.nih.gov/BLAST

R. Chandra et al. / Bioresource Technology 102 (2011) 2333–2341 2335

2.4. Characterization and identification of organic pollutants and theirmetabolites

2.4.1. Isolation of organic pollutants and their metabolitesFor the isolation of organic pollutants and their metabolic prod-

ucts, the bacteria treated and untreated tannery wastewater sam-ples were centrifuged at 5000 rpm for 20 min at 4 �C to removebacterial biomass and other suspended particles. The supernatantobtained was acidified to pH 2.0 using 1 N HCl and extracted thricewith the equal volume (100 ml) of ethyl acetate in a separatingfunnel (500 ml) by intermittent shaking (Marco et al., 2007). Theupper organic layer containing organic pollutants/metaboliteswas separated and evaporated to dryness under vacuum at 40 �C.The dried residue obtained was dissolved in 2.0 ml of acetonitrile(HPLC grade), filtered through syringe filter (0.22 lm) and usedfor HPLC analysis.

2.4.2. HPLC analysisThe HPLC analysis of organic pollutants and metabolites was

performed by using a HPLC system (Waters 515, USA) equippedwith reverse phase column C-18 (250 mm � 4.6, particle size5 lm) at 27 �C and 2487 UV/VIS detector via millennium software.The ethyl acetate extract (20 ll) was injected into the HPLC andmonitored at wavelength 224 nm to assess the degradation as wellas characterize the organic pollutants and their metabolites pro-duced during the bacterial treatment of tannery wastewater in aer-ation lagoons at CETP. The mobile phase was consisted ofacetonitrile and water in the volume ratio of 70:30 (v/v) and flowrate was set at the rate of 1.0 ml min�1.

2.4.3. GC–MS analysisIn GC–MS analysis, the ethyl acetate extracts were analyzed as

trimethyl silyl (TMS) derivatives as described (Minuti et al., 2006).In this method, 100 ll dioxane and 10 ll pyridine was added tosamples followed by silylation with 50 ll trimethyl silyl [BSTFA(N, O-bis (trimethylsilyl) trifluoroacetamide) and TMCS (trimethylchlorosilane)]. The mixture was heated at 60 �C for 15 min withperiodic shaking to dissolve residues. An aliquot (1 ll) of silylatedsamples were injected in GC–MS (PerkinElmer, UK) equipped witha PE auto system XL gas chromatograph interfaced with a Turbo-mass mass spectrometric mass selective detector. The analyticalcolumn connected to system was a PE-5MS capillary column(20 m � 0.18 mm internal diameter, 0.18 mm film thickness).Helium gas was used as carrier gas with flow rate of 1 ml min�1.The column temperature was programmed as 50 �C (5 min); 50–300 �C (10 �C min�1, hold time: 5 min). The transfer line and ionsource temperatures were maintained at 200 and 250 �C, respec-tively. A solvent delay of 3.0 min was selected. In full-scan mode,the electron ionization (EI) mass spectra were recorded in rangeof 30–550 (m/z) at 70 eV. The organic pollutants and metabolicproducts were identified by comparing their mass spectra withthat of National Institute of Standards and Technology (NIST) li-brary available with instrument and by comparing the retentiontime with those of available authentic organic compounds.

2.5. Toxicity evaluation of tannery wastewater before and afterbacterial treatment

2.5.1. Preparation of bacteria treated and untreated tannerywastewater samples for seed germination experiment

The tannery wastewater collected from CETP before and afterbacterial treatment was centrifuged at 5000�g for 20 min to re-move bacterial biomass and other suspended particles and leftover night to settle down the remaining suspended particles. Ifgrowth occurs, the wastewater samples were centrifuged againto remove it, stored in screw-caped glass bottle and used in seed

germination experiment for the toxicity evaluation of tannerywastewater before and after bacterial treatment in aerationlagoons of CETP.

For seed germination experiment, ten seeds of Phaseolus mungoL. were placed in sterilized glass petri dishes of uniform size linedwith two Whatman 52 filter paper discs (Whatman, England).These filter discs were then moistened with 5 ml of tap water forcontrol and with the same volume of untreated and treated tan-nery wastewater samples followed by incubation at 28 �C in aBOD incubator (Labco, India) for a period of six consecutive daysto record the percent germination and seedling growth (i.e. shootlength and root length). The experiment was performed in tripli-cate. Before sowing, the seeds were surface sterilized with 2.0%HgCl2 solution for 2 min to avoid any fungal contamination (Ahsanet al., 2007) and then washed thrice with double distilled water.

The seeds that germinated were counted and removed frompetri dishes at the time of first count on each day until there wasno further germination. The criterion of seed germination whichwe have taken was visible protrusion of radical from seed coat.The germination rate was determined for seeds kept at ambienttemperature at every 24 h of incubation period for six days. Thegerminated seeds were counted to the initial appearance of radicalby continuous visual observation for six days. The germination in-dex (GI) was calculated according to the method of Ana et al.(2004).

3. Results and discussion

3.1. Bacterial diversity in two aeration lagoons of CETP

The restriction digestion of PCR amplified 16S rRNA gene frag-ment derived from bacterial communities growing in aeration la-goons of CETP with two restriction endonucleases TaqI andSau3AI (Bangalore Genei, India), separately and together has indi-cated the presence of nine bacterial species in aeration lagoon I(Fig. 1A) and six bacterial species in aeration lagoon II (Fig. 1B) interms of differences in unique RFLPs. The restriction digestion of16S rRNA fragments with TaqI and Sau3AI, separately and togetherallowed the separation of total bacterial communities growing inaeration lagoon I and II into four and two distinct groups, respec-tively with each group having a characteristic 16S rRNA RFLPpattern.

In case of the bacterial communities growing in aeration lagoonI, group-I included three bacterial species, all having similar 16SrRNA RFLP patters with fragment size 1507, 1506, and 1499 bp.Group-II has four bacterial species with fragment size 1493,1500, 1505, and 1497 bp whereas group-III and IV has only a singlebacterial species with fragment size 1514 and 1508 bp, respec-tively. But, in case of the bacterial communities growing in aera-tion lagoon II, group-I included five bacterial species with same16S rRNA RFLP pattern consisting the fragments of 1510, 1467,1509, and 1417 bp, whereas group II has only a single bacteriumwith fragment size 1509 bp, respectively.

The partial sequences obtained from the selected bands wereblasted at NCBI data search suggesting the closest homologue ofeach partial sequence. On the basis of percentage similarity, thepartial sequences obtained from the bacterial species of group-Igrowing in aeration lagoon-I have shown the closest relatednesswith that of Escherichia strains whereas that of four bacterialspecies of group II have shown the closest relatedness withStenotrophomonas strains. Similarly, the sequences obtained fromthe bacterial species belonging to group III and IV has shown theclosest homology with that of Bacillus sp. and Cronobacter sp.,respectively. These two groups (i.e. I and II) have represented themajor portion (i.e. 33.33 and 44.44%) of the total bacterial diversity

Fig. 1A. RFLP profile generated by the restriction digestion of 16S rRNA genefragment derived from bacterial community growing in aeration lagoon-I by usingTaq I (a), Sau3AI (b) and Taq I and Sau3AI endonuclease together (c). The abovepictures shows the clones, labeled from T1–T37 (a), S18–S48 (b) and from S1–S17(c) which are PCR positive and subjected to sequencing analysis. L1-100 bp ladder;L2-500 bp ladder.

Fig. 1B. RFLP profile generated by the restriction digestion of 16S rRNA genefragment derived from bacterial community growing in aeration lagoon-II by usingTaq I (a), Sau3AI (b) and Taq I and Sau3AI endonuclease together (c). The abovepictures shows the clones, labeled from T1–T41 (a), S24–S59 (b) and from T43–T59(c) which are PCR positive and subjected to sequencing analysis. L1-100 bp ladder;L2-500 bp ladder.


growing in aeration lagoon-I and belong to c-proteobacteriawhereas groups III and IV have represented 11% of the total bacte-rial diversity growing in aeration lagoon-I and shown the closestrelatedness with that of Cronobacter sp. and Bacillus sp. belongingto enterobacteriales and firmicutes, respectively (Fig. 2A). Hence,on the basis of sequence homology, it is clear that groups I–IV ofaeration lagoon-I is dominated by Escherichia, Stenotrophomonas,Bacillus and Cronobacter sp., respectively.

Moreover, the bacterial communities growing in aeration la-goon-I was somewhat similar to that of aeration lagoon-II. In aer-ation lagoon-II, the five bacterial species of group I and singlebacterial species of group II have shown the closest relatednesswith that of Stenotrophomonas sp. and Burkholderiales bacterium,respectively. Group I represented 83.33% of the total bacterialdiversity growing in aeration lagoon-II whereas group II has repre-sented 16.66% of the total bacterial diversity growing in aerationlagoon-II that belongs to c and b-proteobacteria, respectively(Fig. 2B). Further, the partial sequences of bacterial diversity grow-

ing in aeration lagoon-I and II were deposited to GenBank publicdatabase under the accession number FJ422465–FJ422473 foraeration lagoon-I and from FJ422474–FJ422479 for aerationlagoon-II, respectively.

The phylogenetic tree of bacterial communities growing inaeration lagoon-I contained five Escherichia coli strains, threeCronobacter strains, six Stenotrophomonas strains, three Bacillussp. (firmicutes), and one–one strain of Pseudomonas sp., Acinetobac-ter sp., Ralstonia sp. (b-proteobacteria), Sphingomonas sp. (a-proteo-bacteria) and a single strain of Methanobacterium (archaea),respectively. These bacterial strains have shown closest related-ness with that of present in aeration lagoon-I. However, the phylo-genetic tree of bacterial diversity growing in aeration lagoon-IIincluded seven Stenotrophomonas sp. belongs to c-proteobacteria,and single strains of Burkholderiales sp., Ralstonia sp., Pseudomonassp., Acinetobacter sp., Sphingomonas sp. and Methanobacteriumbelonging to b-proteobacteria, c-proteobacteria, a-proteobacteriaand archaea group, respectively. All these bacterial species haveshown the closest similarity with that bacterial species dominatingin aeration lagoon-II of CETP.

B

0.1

Ecoli

Ecoli2996

Cronobacter

Crono982

519

T1IITR9

T1IITR5421

T1IITR6

1000

1000

T1IITR8

491

T1IITR4

T1IITR3708

T1IITR1

998

StenoM

Steno914

1000

T1IITR2

465

483

X96787 P

Z93447884

355

AF312022 R

1000

AF503283

998

AJ577290 B

AF290547 B1000

T1IITR7

1000

AY196659

Escherichia coli strain ATCC 11229 [GQ340751]

Escherichia coli strain ATCC25922 [DQ683069 ]

Cronobacter sakazakii strain E750; ATCC 29004 [EF059868]

Cronobacter muytjensii strain E603; ATCC 51329 [EF059845 ]

Escherichia coli strain T1IITR9 [FJ422473]



Cronobacter sp. strain T1IITR8 [FJ422472]

Stenotrophomonas sp. strain T1IITR4 [FJ422468]



Stenotrophomonas maltophilia strain ATCC 13637 [FJ971878]

Stenotrophomonas acidaminiphila strain AMX19; ATCC 700916 [NR025104]


Pseudomonas multiresinivorans strain ATCC 700690T [X96787]

Acinetobacter sp.strain ATCC 17905 [Z93447 ]

Ralstonia basilensis strain DSM 11853 [AF312022]

Sphingomonas sp. ATCC No.53159 [AF503283 ]

Bacillus cereus strain ATCC 10987 [AJ577290 ]

Bacillus sp. strain ATCC14579 [AF290547 ]

Bacillus sp. strain T1IITR7 [FJ422471]

Methanobacteriumformicicum [AY196659 ]0.1

Ecoli

Ecoli2996

Cronobacter

Crono982

519

T1IITR9

T1IITR5421

T1IITR6

1000

1000

T1IITR8

491

T1IITR4

T1IITR3708

T1IITR1

998

StenoM

Steno914

1000

T1IITR2

465

483

X96787 P

Z93447884

355

AF312022 R

1000

AF503283

998

AJ577290 B

AF290547 B1000

T1IITR7

1000

AY196659

Escherichia coli strain ATCC 11229 [GQ340751]

Escherichia coli strain ATCC25922 [DQ683069 ]

Cronobacter sakazakii strain E750; ATCC 29004 [EF059868]

Cronobacter muytjensii strain E603; ATCC 51329 [EF059845 ]




Cronobacter sp. strain T1IITR8 [FJ422472]




Stenotrophomonas maltophilia strain ATCC 13637 [FJ971878]



Pseudomonas multiresinivorans strain ATCC 700690T [X96787]

Acinetobacter sp.strain ATCC 17905 [Z93447 ]

Ralstonia basilensis strain DSM 11853 [AF312022]


Bacillus cereus strain ATCC 10987 [AJ577290 ]

Bacillus sp. strain ATCC14579 [AF290547 ]

Bacillus sp. strain T1IITR7 [FJ422471]

Methanobacteriumformicicum [AY196659 ]

γ-proteobacteria

Enterobacteriales

β-proteobacteria

α-proteobacteria

Firmicutes

Archaea

A

Fig. 2A. Phylogenetic tree showing the relationship of bacterial communities growing in aeration lagoon I with bacteria of different taxa. The accession numbers for all strainsused in tree construction are indicated in the figure in square brackets.


3.2. Physico-chemical characteristics of tannery wastewater

The physico-chemical analysis of tannery wastewater beforebacterial treatment has revealed that it has high BOD, COD values,TS, sulfate, phosphate, organic pollutants and heavy metals (Cr, Cu,Zn, Fe, Ni, Mn and Pb) Table 1. But, bacterial treatment in aerationlagoon I and II has resulted 92, 86.87, 89.75, 58.13, 54.58, 70.49,and 77.02% reduction in BOD, COD, TS, TN, sulfate, phosphate,and organic pollutants, respectively (Table 1). The bacterial com-munities growing in aeration lagoon I and II were also found highlyeffective to reduce 87.96, 75.45, 73.71, and 86.2% Cr, Zn, Fe, andMn, respectively. The reduction in metals content might be occureither through metal bioaccumulation inside cells or binding withlipopolysaccharides of extra cellular membrane (Srinath et al.,2002). The reduction in BOD, COD and TS might be attributed tothe bacterial degradation of complex organic and inorganic pollu-tants to meet the nutritional requirements.

3.3. Bacterial mechanism of Cr6+ detoxification/removal in aerationlagoons at CETP

During wastewater treatment in aeration lagoons at CETP, theprocesses by which microbes interact with metals enabling their

removal/and recovery are biosorption, bioaccumulation andenzymatic reduction. Biosorption is a metabolism-independentprocess, which largely depends on mechanisms such as complexa-tion, ion exchange, coordination, adsorption, chelation andmicroprecipitation and can be performed by both living and deadcells (Srinath et al., 2002; Roane et al., 2001). In some microbes likeZoogloea ramigera, Klebsiella aerogenes, Arthrobacter viscosus andPseudomonas sp., the exopolymers of capsule/slime layer are themajor site of metal biosorption. The Cr6+ uptake is mediated by‘‘acid adsorption’’ mechanism in which liquid should have enoughprotons to cause anion exchange and after accumulation, Cr6+ mayact as terminal electron acceptor and reduced Cr3+ then binds tocell wall (Cabrera et al., 2007; Srinath et al., 2002).

The microbial heavy metal accumulation comprised twophases: an initial rapid phase involving physical adsorption orion exchange at cell surface and by a subsequent slower phaseinvolving active metabolism-dependent transport of Cr6+ into thebacterial cells. During the bioaccumulation process, many featuresof a living cell like intracellular sequestration followed by localiza-tion within specific organelles, metallothionein binding, particu-late metal accumulation, extracellular precipitation and complexformation can occur (Shukla et al., 2009; Cabrera et al., 2007;Srivastava et al., 2007). Recently, heavy metals accumulating

γ-proteobacteria

γ-proteobacteria

β-proteobacteria

α-proteobacteria

Archaea0.1

T1IITR10

T1IITR12

990

T1IITR14

630

T1IITR11

T1IITR13

1000

913

Steno

434

StenoM

1000

T1IITR15

AF312022 R

1000

586

X96787 P

Z93447

855

999

AF503283

AY196659

Stenotrophomonas sp. T1IITR10 [FJ422474]






Stenotrophomonas maltophilia strain ATCC 13637 [FJ971878 ]

Burkholderiales sp. T1IITR15 [FJ422479]

Ralstonia basilensis strain DSM 11853 [AF312022 ]

Pseudomonas multiresinivorans strainATCC 700690T [X96787]

Acinetobacter sp. strain ATCC 17905 [Z93447 ]


Methanobacterium formicicum[AY196659 ]

0.1

T1IITR10

T1IITR12

990

T1IITR14

630

T1IITR11

T1IITR13

1000

913

Steno

434

StenoM

1000

T1IITR15

AF312022 R

1000

586

X96787 P

Z93447

855

999

AF503283

AY196659







Stenotrophomonas maltophilia strain ATCC 13637 [FJ971878 ]

Burkholderiales sp. T1IITR15 [FJ422479]

Ralstonia basilensis strain DSM 11853 [AF312022 ]

Pseudomonas multiresinivorans strainATCC 700690T [X96787]

Acinetobacter sp. strain ATCC 17905 [Z93447 ]


Methanobacterium formicicum[AY196659 ]

B

Fig. 2B. Phylogenetic tree showing the relationship of bacterial communities growing in aeration lagoon II with bacteria of different taxa. The accession numbers for allstrains used in tree construction are indicated in the figure in square brackets.

Table 1Physico-chemical characteristics of tannery wastewater before and after bacterial treatment in aeration lagoons of CETP.

Physico-chemical parameters Value (s) in Tannery effluent

Untreated Aeration lagoon-I % Reduction in pollutants Aeration lagoon-II % Reduction in pollutants

pH 8.5 ± 0.17 7.5 ± 0.20 – 7.2 ± 0.15 –BOD 2933 ± 126 1066 ± 110 63.65 236 ± 53 92COD 12,466 ± 321 5712 ± 136 54.17 1636 ± 110 86.87TS 60,180 ± 246 18,680 ± 238 68.95 6170 ± 112 89.75TN 578 ± 63 363 ± 46 37.19 242 ± 23 58.13SO4

– 6.65 ± 1.12 4.33 ± 1.10 34.88 3.02 ± 1.02 54.58PO4

— 427 ± 87 265 ± 32 48.47 126 ± 16 70.49Phenolics 1.48 ± 0.23 0.52 ± 0.06 64.86 0.34 ± 0.02 77.02

Heavy metalsCr 38.90 ± 13.16 11.05 ± 1.03 71.59 4.68 ± 0.20 87.96Cu 0.18 ± 0.02 ND – ND -Zn 1.10 ± 0.20 0.63 ± 0.05 42.72 0. 27 ± 0.03 75.45Fe 7.57 ± 1.23 4.69 ± 1.52 38.04 1.99 ± 0.24 73.71Ni 1.38 ± 0.23 ND – ND –Mn 0.58 ± 0.04 0.17 ± 0.03 70.68 0.08 ± 0.02 86.2Pb 0.09 ± 0.04 ND – ND –

All the values are in (mg l�1) and means of three replicate (n = 3) ± SD. BOD: Biological oxygen demand; COD: Chemical oxygen demand; TS: Total solids; TN: Total nitrogen;ND: Not detectable.


bacterial strains have sought a new role as ‘small factories’ for pro-duction of nanoparticles (Cheung and Gu, 2007).

3.3.1. Enzyme mediated aerobic and anaerobic reduction of Cr6+ toCr3+

A number of bacterial species such as Bacillus sp., Pseudomonassp., Alcaligenes sp., E. coli, and Shewanella alga are reported to have

Cr6+ detoxification capability due to presence of reductases solublein cytosol. In Pseudomonas maltophilia O�2 and Bacillus megateriumTKW3, Cr6+ reduction is associated with membrane cell fractions(Shukla et al., 2009; Cabrera et al., 2007; Srinath et al., 2002). Dur-ing bacterial treatment of tannery wastewater in aeration lagoonsat CETP, Cr6+ reduction commonly occurs in two or three steps inwhich Cr6+ initially reduced to a short-lived intermediates Cr5+


and/or Cr4+ before further reduction to the thermodynamically sta-ble end product Cr3+ (Cheung and Gu, 2007). However, at present itis unclear that whether the reduction of Cr5+ to Cr4+ and Cr4+ to Cr3+

is spontaneous or enzyme mediated. The NADH, NADPH and elec-trons from the endogenous reservoir are implicated as electron do-nors in Cr6+ reduction process. During the reduction process, theenzyme Cr6+ reductase (ChrR) transiently reduces Cr6+ with aone-electron shuttle to form Cr5+ followed by a two-electron trans-fer to generate Cr3+ (Ackerley et al., 2004). Although a proportion ofthe Cr5+ intermediate is spontaneously reoxidized to generate reac-tive oxygen species (ROS), its reduction through two-electrontransfer catalyzed by ChrR reduces the opportunity to produceharmful radicals.

Moreover, another enzyme YieF is unique in that it catalyzes thedirect reduction of Cr6+ to Cr3+ through a four-electron transfer, inwhich three electrons are consumed in reducing Cr6+ and the otheris transferred to oxygen. Since the quantity of ROS generated byYieF in Cr6+ reduction is minimal, it is regarded as a more effectivereductase than ChrR for Cr6+ reduction (Cabrera et al., 2007;Cheung and Gu, 2007). The membrane-associated Cr6+ reductaserecently isolated from B. megaterium TKW3 utilized NADH as anelectron donor, but the kinetics of Cr6+ reduction is not yet known.Park et al. (2000) purified 600-fold a soluble Cr6+ reductase, ChrR,from Ps. Putida MK1. The ChrR-coding gene, chrR, was identifiedfrom the genomic sequence of Ps. putida MK1, based on the knownamino acid sequences of the N-terminal and internal amino acidsegments of the pure enzyme. Latter, Ackerley et al. (2004) de-scribed ChrR as a dimeric flavoprotein catalyzing the reduction ofCr6+ optimally at 70 �C. An open reading frame, yieF, on theE. coli chromosome with no assigned function was found to havea high homology with chrR. This gene was cloned and the encodedprotein, YieF, showed maximum reduction of Cr6+ at 35 �C.Recently, the gene encoding this reductases enzyme was foundto exhibit a high nucleotide sequence homology (58%) to a nitrore-ductase of Vibrio harveyi KCTC 2720 that was also endowed withCr6+ reducing activities (Cheung and Gu, 2007).

On the other hand, several facultative anaerobes such as Ps.dechromaticans, Ps. Chromatophila, Aeromonas dechromatica,Microbacterium sp. MP30, Geobacter metallireducens, Shewanellaputrefaciens MR-1, Pantoea agglomerans SP1, and Agrobacteriumradiobacter EPS-916 are also reported to catalyze the biotransfor-mation of Cr6+ to Cr3+ under anoxic conditions. But, unlike to Cr6+

reductases isolated from aerobes, the Cr6+ reducing activities ofanaerobes are associated with their electron transfer systems ubiq-uitously catalyzing the electron shuttle along with the respiratorychains (Cheung and Gu, 2007; Cabrera et al., 2007; Srinath et al.,2002). In anaerobes, both soluble and membrane-associated en-zymes were found to mediate the Cr6+ reduction process underanaerobic conditions. Moreover, the natural metabolites of anaer-obes such as H2S produced by sulfate-reducing bacteria (SRB) areconsidered as an important agent for Cr6+ reduction under anoxicenvironment. In past, the anaerobic reduction of Cr6+ was consid-ered as a fortuitous process that provides no energy for microbialgrowth. But, recently a SRB isolate was found to use the energygenerated during the anaerobic Cr6+ reduction for its growth(Shukla et al., 2009; Moosvi and Madamwar, 2007). In absence ofoxygen, Cr6+ can also serve as a terminal electron acceptor in respi-ratory chain for a large array of electron donors, including carbohy-drates, proteins, fats, hydrogen, NAD (P) H and endogenouselectron reserves (Cheung and Gu, 2007).

In Desulfomicrobium norvegicum, a hydrogenase and a C-typecytochrome was found to be involved in Cr6+ reduction mechanismwhereas Desulfotomaculum reducens MI-1 was found capable ofutilizing Cr6+ as sole electron acceptor. The cytochrome families(e.g., cytochrome b and cytochrome c) were frequently shown tobe involved in the anaerobic enzymatic Cr6+ reduction process

(Moosvi and Madamwar, 2007; Cheung and Gu, 2007; Srinathet al., 2002). The widespread occurrence of anaerobes possessingCr6+ reducing capabilities offers great potential for in situ bioreme-diation of Cr6+ contaminated environment/sediments, whichwould only require the supplementation of nutrients and the mod-ulation of physical conditions to facilitate the bioremediation ofCr6+ from contaminated environment/sediments.

3.4. Characteristics of organic pollutants and their metabolites

The degradation of organic pollutants and their concentrationduring the bacterial treatment of tannery wastewater in aerationlagoon I and II was monitored by HPLC analysis, which showedfast/drastic reduction in pollutants concentration during the bacte-rial treatment process in terms of reduction in peak area for aera-tion lagoon I and II compared to untreated tannery wastewater.The reduction in peak areas has clearly indicated the degradationof organic pollutants during the bacterial treatment process of tan-nery wastewater in aeration lagoon I and II. In addition, there weresome additional peaks observed in bacterial treated samples sug-gesting the formation of some new pollutants/metabolites thatwere further confirmed by the GC–MS analysis. Reduction in peakareas and generation of some new peaks has clearly indicated thebiodegradation as well as biotransformation of organic pollutantsduring the bacterial treatment of tannery wastewater in aerationlagoon I and II at CETP.

Further, the GC–MS analysis of untreated tannery wastewatersamples showed the presence of various types of organic pollu-tants (Table 2), most of which were biodegraded during bacterialtreatment process in aeration lagoon I and II. The analysis of bacte-ria treated tannery wastewater samples has showed the presenceof diethylene glycol, ethanol, cyclohexane, octadecanoic acid,hexadecanoic acid, 2-(1-methyl-1,2,3,4-tetrahydro-isoquinolin-1-yl)-butanol-2, 3-hydroxy propanoic acid, 2-butenoic acid, and 2-oxovaleric acid in aeration lagoon I of CETP. During the bacterialtreatment of tannery wastewater in aeration lagoon I and II, twopollutants i.e. L-(+)-lactic acid and acetic acid could not be metab-olized whereas benzene and 2-hydroxy-3-methyl-butanoic acidwas produced as new metabolites in aeration lagoon II of CETP.However, most of the organic pollutants detected in untreatedtannery wastewater samples were diminished from bacteriatreated tannery wastewater samples. The disappearance of mostof organic pollutants from tannery wastewater collected fromaeration lagoon I and II has revealed that bacterial communitiesgrowing in aeration lagoon I and II utilized these organic pollutantsas sole source of C, N, and energy and play a major role in thedegradation and detoxification of tannery wastewater for environ-mental safety.

3.5. Effect of bacteria treated and untreated tannery wastewater onseed germination and seedling growth

The physico-chemical analysis has revealed that untreatedtannery wastewater was highly toxic in nature and has inhibitoryeffect on seed germination and seedling growth. But, after bacterialtreatment in aeration lagoon I and II at CETP, its toxicity was re-duced significantly and has shown improved seed germinationcompared to untreated tannery wastewater. However, various cropplant species differed widely in response to different industrialwastewaters with respect to seeds germination, seedling growthand productivity (Ramana et al., 2002). In present study, it was ob-served that untreated tannery wastewater was highly toxic in nat-ure and showed only 40% seed germination but after bacterialtreatment in aeration lagoon I and II, the toxicity of tannery waste-water was reduced significantly and showed 70% and 90% seed ger-mination, respectively (Table 3).

Table 2Organic pollutants and their metabolites identified as TMS (trimethyl silyl) derivatives by GC–MS analysis in tannery wastewater before and after bacterial treatment at CETP.

Sr. No. Compounds identified RT Tannery wastewater

Untreated Aeration lagoon I Aeration lagoon II

1. 2-methylbutanoic acid 8.9 + � �2. Pentanoic acid 9.3 + � �3. L-(+)-Lactic acid 9.4 + + +4. Hexanoic acid 9.8 + � �5. 4-methylvaleric acid 9.9 + � �6. Propanoic acid 10.0 + � �7. Acetic acid 10.2 + + +8. Butanedioic acid 10.5 + � �9. Benzene acetic acid 11.1 + � �10. 3-methylbutanoic acid 11.4 + � �11. Benzoic acid 12.6 + � �12. Decanoic acid 13.7 + � �13. Benzene propanoic acid 15.5 + + �14. 1,1-dimethylethyl-2-phenylethiazole 16.9 + � �15. Cyclobutane 17.0 + � �16. 3-methoxy-4-benzaldehyde 17.6 + � �17. Tartaric acid 18.4 + � �18. Acetylthiocarbamic acid 20.27 + � �19. Diethylene glycol 9.4 � + �20. Ethanol 10.52 � + �21. 2-(1-methyl-1,2,3,4-tetrahydro-isoquinolin-1-yl)-butan-2-ol 9.75 � + �22. 3-hydroxy propanoic acid 10.5 � + -23. 2-butenoic acid 14.79 � + �24. 2-oxovaleric acid 16.04 � + �25. Benzene 8.8 � � +26. 2-hydroxy-3-methyl-butanoic acid 8.9 � � +

+: present; �: absent; TMS: trimethyl silyl.

Table 3Effect of untreated and treated tannery wastewater on seed germination and seedling growth in Phaseolus mungo L.

Tannery effluent No. of seeds sown % Germination Speed of germination index Shoot length (cm) Root length (cm) Root/shoot ratio

Untreated 10 40 92 ± 2.11 0.52 ± 0.03 0.00 ± 0.00 0.00 ± 0.00Treated (A-I) 10 70 130 ± 2.84 1.68 ± 0.28 0.78 ± 0.12 0.46 ± 0.04Treated (A-II) 10 90 212 ± 4.87 3.56 ± 0.42 2.26 ± 0.36 0.63 ± 0.06Control (TW) 10 100 230 ± 6.34 4.84 ± 0.76 3.46 ± 0.34 0.71 ± 0.05

A-I: Aeration lagoon-I; A-II: Aeration lagoon-II, TW: Tap Water.


However, seed germination is a complex physiological and bio-chemical process that can be affected by several environmentalfactors. Starch is the major component of most of the world’s cropyield and degradation of starch is essential for seed germination. Ingerminating seeds, starch degradation is initiated by a-amylase(Ahsan et al., 2007) producing soluble oligosaccharides fromstarch. These are then hydrolyzed by b-amylase to liberate maltoseand finally, a-glucosidase breaks down maltose into glucose pro-viding energy to germinating seeds. The inhibitory effect of un-treated tannery wastewater on seed germination might be due tothe presence of high content of total solids, total nitrogen, phos-phate, sulfates, and heavy metals, which induces high osmoticpressure and anaerobic conditions. This high osmotic pressureand anaerobic conditions render various physiological andbiochemical processes such as movement of solute, respirationprocess of seeds and enzymatic steps of seed germination.

Further, regarding the seedling growth (i.e. shoot lengthand root length), the present study has revealed that the seeds ex-posed to untreated tannery wastewater showed no root develop-ment and very short shoot (0.52 cm). But, the seeds exposed totreated tannery wastewater showed the development of bothshoot and root. The seeds exposed to tannery wastewater treatedin aeration lagoon I showed reduced root length (0.78 cm) andshoot length (1.68 cm) compared to seeds exposed to tannerywastewater treated in aeration lagoon II (root length: 2.26 cm;shoot length: 3.56 cm). The inhibitory effect of untreated tannery

wastewater on seed germination and seedling growth might beattributed to the high salt load and metals content, which induceshigh osmotic pressure and anaerobic conditions (Ramana et al.,2002). This high osmotic pressure and anaerobic conditions rendervarious physiological and biochemical processes of seed germina-tion and seedling growth such as movement of solute, respirationprocess of seeds and enzymatic steps of seed germination andseedling growth. Further, it has been reported that high salt loadand metals content act as inhibitor for plant hormones (s) (amy-lases, auxins, gibberlines and cytokinins), which are mainly re-quired for seed germination, seedling growth and developmentof plants, respectively (Ahsan et al., 2007). The deleterious effectsof tannery wastewater on plant growth parameters might be alsodue to the entrance of toxic heavy metals into the protoplasmresulting in the loss of intermediate metabolites, which are essen-tial for further growth and development of plants as chromium isspecifically reported to have inhibitory effect on root growth morethan on coleoptiles growth prior to germination of seeds (Ahsanet al., 2007).

4. Conclusions

In this study, the 16S rRNA gene sequence analysis has revealedthat aeration lagoon I was highly rich in bacterial diversitycontaining Escherichia sp., Stenotrophomonas sp., Bacillus sp., andCronobacter sp., whereas aeration lagoon II was dominated by


Stenotrophomonas sp., and Burkholderiales bacterium, respectively.Theses bacterial species have been already reported by variousauthors to play an important role in the degradation and detoxifi-cation of tannery wastewater. However, the physico-chemicalanalysis of tannery wastewater collected from aeration lagoon IIafter bacterial treatment has shown 92, 86.87, 89.75, 58.13,54.58, 70.49, and 77.02% reduction in BOD, COD, TS, TN, sulfate,phosphate, and organic pollutants, respectively. Further, the HPLCand GC–MS analysis showed that L-(+)-lactic acid and acetic acidcould not be degraded by bacteria whereas benzene and 2-hydro-xy-3-methyl-butanoic acid was produced as new metabolites dur-ing the bacterial treatment in aeration lagoon II at CETP. Inaddition, it was also observed that the toxicity of tannery wastewa-ter was reduced significantly after bacterial treatment process inaeration lagoon I and II at CETP and showed 70% and 90% seed ger-mination, respectively.

Acknowledgements

The financial assistance to Mr. R.N. Bharagava, SRF from Univer-sity Grants Commission and Council of Scientific and IndustrialResearch (CSIR-Network Project NWP-19) New Delhi is highlyacknowledged. We also acknowledge the support of Dr. SubhashAwasthi, In-charge CETP, Unnao for providing the samples of differ-ent stage from CETP.

References

Ackerley, D.F., Gonzalez, C.F., Keyhan, M., Blake II, R., Matin, A., 2004. Mechanism ofchromate reduction by the E. Coli protein, NfsA, and the role of differentchromate reductases in minimizing oxidative stress during chromate reduction.Environmental Microbiology 6, 851–860.

Ahsan, N., Lee, S.H., Lee, D.G., Lee, H., Lee, S.W., Bahk, J.D., Lee, B.H., 2007.Physiological and protein profiles alternation of germinating rice seedlingsexposed to acute cadmium toxicity. Comptes Rendus Biologies 330, 735–746.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.,1997. Gapped BLAST and PSIBLAST: a new generation of protein databasesearch programs. Nucleic Acids Research 25, 3389–3402.

Ana, F., Merceds, L., Jose, S., Isabel, A.M., Juan, F.O., Victor, F.M., 2004. Phytotoxicityand heavy metals speciation of stabilized sewage sludge. Journal of HazardousMaterials 108, 161–169.

APHA, 2005. Standard Methods for the Examination of Water and Waste Water.seven-teenth ed. American Public Health Association. Washington, DC.

Baker, C.J., Fulthorpe, R.R., Gilbride, K.A., 2003. An assessment of variability of pulpmill wastewater treatment system bacterial communities using molecularmethods. Water Quality Research Journal of Canada 38, 227–242.

Cabrera, G., Viera, M., Gomez, J.M., Cantero, D., Donati, E., 2007. Bacterial removal ofchromium (VI) and (III) in a continuous system. Biodegradation 18, 505–513.

Cheung, K.H., Gu, J.D., 2007. Mechanism of hexavalent chromium detoxification bymicroorganisms and bioremediation application potential: a review.International Biodeterioration & Biodegradation 59 (1), 8–15.

Coelho, M.R.R., Weid, I.V.D., Zahner, V., Seldin, L., 2003. Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragmentlength polymorphism analysis of part of genes encoding 16S rRNA and 23S

rRNA and by multilocus enzyme electrophoresis. FEMS Microbiology Letters222, 243–250.

Henriques, I., Alves, A., Tacao, M., Almeida, A., Cunha, A., Correia, A., 2006. Seasonaland spatial variability of free-living bacterial diversity composition along anestuarine gradient (Ria de Aveiro, Portugal). Estuarine Coastal Shelf Science 68,139–148.

Kapley, A., De Baere, T., Purohit, H.J., 2007. Eubacterial diversity of activatedbiomass from a common wastewater treatment plant. Research in Microbiology158, 494–500.

Lane, D.J., 1991. 16S/23S rRNA Sequencing. In: Stackebrandt, F., Goodfellow, M.(Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons,New York, pp. 115–175.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam,H.C.W., 2007. Clustal X version 2.0. Bioinformatics 23, 2947–2948.

Marco, E.D., Savarese, M., Paduano, A., Sacchi, R., 2007. Characterization andfractionation of phenolic pollutants extracted from olive oil mill wastewaters.Food Chemistry 104, 858–867.

Minuti, L., Pellegrino, R.M., Tesei, I., 2006. Simple extraction method and gaschromatography–mass spectrometry in the selective ion monitoring mode forthe determination of phenols in wine. Journal of Chromatography – A 1114,263–268.

Moharikar, A., Kumar, R., Purohit, H.J., 2005. Microbial population dynamic atwastewater treatment plants. Journal of Environmental Monitoring 7, 552–558.

Moosvi, S., Madamwar, D., 2007. An integrated process for the treatment of CETPwastewater using coagulation, anaerobic and aerobic process. BioresourceTechnology 98, 3384–3392.

Moura, A., Tacao, M., Henriques, I., Dias, J., Ferreira, P., Correia, A., 2009.Characterization of bacterial diversity in two aeration lagoons of awastewater treatment plant using PCR–DGGE analysis. MicrobiologicalResearch 164 (5), 560–569.

Munz, G., Gualtiero, M., Salvadori, L., Claudia, B., Claudio, L., 2008. Process efficiencyand microbial monitoring in MBR (membrane bioreactor) and CASP(conventional activated sludge process) treatment of tannery wastewater.Bioresource Technology 99, 8559–8564.

Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S., Matin, A., 2000. Purification tohomogeneity and characterization of a novel Pseudomonas putida chromatereductase. Applied and Environmental Microbiology 66, 1788–1795.

Ramana, S., Biswas, A.K., Singh, A.B., Yadav, R.B.R., 2002. Relative efficacy of differentdistillery wastewaters on growth, nitrogen fixation and yield of groundnut.Bioresearch Technology 81 (2), 117–121.

Roane, T.M., Josephson, K.L., Peppar, I.L., 2001. Dual bioaugmenation strategy toenhance remediation of contaminated soil. Applied and EnvironmentalMicrobiology 67, 3208–3215.

Shi-Yan, Y., Xiao-Yan, Z., Du, X., Tai-Ming, Z., Yong-Ming, L., San-Jun, C., Xiao-Li, X.,Bao-Hua, Y., Heng-Hua, Z., Da-Ren, S., 2007. Three novel missense germlinemutations in different exons of MSH6 gene in Chinese hereditary non-polyposiscolorectal cancer families. World Journal of Gastroenterology 13 (37), 5021–5024.

Shukla, O.P., Rai, U.N., Dubey, S., 2009. Involvement and interaction of microbialcommunities in the transformation and stabilization of chromium during thecomposting of tannery effluent treated biomass of Vallisneria spiralis L.Bioresource Technology 100, 2198–2203.

Srinath, T., Verma, T., Ramteke, P.W., Garg, S.K., 2002. Chromium (VI) biosorptionand bioaccumulation by chromate resistant bacteria. Chemosphere 48, 427–435.

Srivastava, S., Ahmad, A.H., Thakur, I.S., 2007. Removal of chromium andpentachlorophenol from tannery wastewaters. Bioresource Technology 98,1128–1132.

Verma, T., Srinath, T., Gadpayle, R.U., Ramteke, P.W., Hans, R.K., Garg, S.K., 2001.Chromate tolerant bacteria isolated from tannery wastewater. BioresourceTechnology 78, 31–35.

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Bacterial diversity, organic pollutants and their metabolites in two aeration lagoons of common effluent treatment plant (CETP) during the degradation and detoxification of tannery